Lhrh-platinum conjugates for treating reproductive cancers

ABSTRACT

The conjugation of luteinizing hormone-releasing hormone (LHRH) with activated cisplatin using a malonate linker gives rise to a new Platinum-LHRH conjugate that effectively targets tumor cells that express the LHRH receptor. The Pt-LHRH conjugate may be used in a method for killing or inhibiting the growth of a tumor cell, especially in late state, highly invasive and aggressive stage IV tumors and in reoccurring tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/159,232 filed Oct. 12, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/367,428 filed Dec. 2, 2016, the disclosures ofwhich are incorporated herein by reference. U.S. patent application Ser.No. 16/159,232 and U.S. patent application Ser. No. 15/367,428 bothclaim the benefit of U.S. Provisional Application No. 62/263,350, filedDec. 4, 2015, the disclosure of which is also incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Award NumberP20GM103436 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

The present invention relates to treating and preventing cancers, moreparticularly to Platinum (Pt)-LHRH conjugates for treating reproductivecancers that allows for the effective delivery of cytotoxic drugs in theplatinum family to a tumor cell.

DISCUSSION OF RELATED ART

Prostate and breast cancer are some of the most common cancers amongindividuals in the United States. Although the incidence of prostatecancer is markedly age dependent and about two-fold higher inAfrican-Americans than Caucasians, prostate cancer is one of the leadingcauses of cancer deaths among men of all races. Conventionalchemotherapeutic approaches yield median overall survival rates forstage IV prostate of only twenty-nine percent (29%) and stage IV breastcancer of only twenty-three percent (23%). Despite recent advances inthe first-line treatment of both cancer types, many patients eventuallyrelapse; their tumors become chemo resistant; leading to patientmortality. One of the problems in cancer chemotherapy is the deleteriousside effects of anticancer drugs designed to destroy rapidly dividingcells, including cells found in healthy tissues. Due to these severeside effects, doctors often resort to dose reduction, treatment delay ordiscontinuance of therapy.

Cisplatin and carboplatin are two known chemotherapeutic drugs in theplatinum (Pt) family of anticancer drugs. They are the most potent drugsin the Pt family and are commonly given in late state or stage IV cancertypes as they effectively disrupt cell cycle/cellular division and cancause cancer cells cytotoxicity/apoptosis leading to tumor death orreduction in size. Cisplatin inhibits growth of tumor cells byinterference with normal transcription and DNA replication mechanismsleading to eventual cell death. Cisplatin chemotherapy, however, is notcurrently very effective in prostate cancer treatment due to its poortargeting of tumor cells and the development of chemo resistant tumors.Carboplatin, an analog of cisplatin, has gained greater use because ofits decreased nonspecific toxicity and its activity againstcisplatin-resistant tumors. See I. Hamelers, et al., “Carboplatinnanocapsules: a highly cytotoxic, phospholipid-based formulation ofcarboplatin,” Molecular Cancer Therapeutics 2006, 5 (8), (2007-12).

Due to their lack of selectivity towards cancer cells the high dosesadministered to a patient to reach a therapeutic response for bothcarboplatin and cisplatin cause severe side effects including: nausea,vomiting, taste changes, hair loss, weakness, abdominal pain, diarrhea,mouth sore, infection, peripheral neuropathy, central neurotoxicityhearing loss, abnormal blood electrolyte levels, abnormal liver enzymelevels, cardiovascular events, dizziness, fatigue, etc. See L. X.Cubeddu, et al., “Efficacy of ondansetron (GR 38032F) and the role ofserotonin in cisplatin-induced nausea and vomiting,” The New EnglandJournal Of Medicine, 322 (12), pp. 810-6 (1990). Additionally, certaincancers can develop resistance to cisplatin via efflux pumps. See K.Katano, et al., “Acquisition of resistance to cisplatin is accompaniedby changes in the cellular pharmacology of copper,” Cancer Research, 62(22), pp. 6559-65 (2002). Despite the current lack of effectivechemotherapies utilizing cisplatin, the drug remains attractive fordevelopment in cancer treatments due to its potency. Accordingly, thedevelopment of treatment methods and chemotherapies that will betterdirect cisplatin to its desired target, tumor cells, with fewer sideeffects and improved efficacy is needed.

Various studies have found that receptors for certain peptides/hormonesare found in higher concentration in tumor cells than in normal cells.For example, the receptor for luteinizing hormone releasing hormone(LHRH) is overexpressed in numerous types of cancer cells including butnot limited to reproductive cancers including breast, ovarian, andprostate cancers. See A. Taheri, “The in-vivo antitumor,” InternationalJournal of Pharmaceutics, (2012). Various non-reproductive cancers havebeen shown to overexpress the LHRH receptor as well, including but notlimited to: lung, bladder, and pancreatic cancers, among others. LHRH,also referred to as gonadotropin releasing hormone (GnRH), is adecapeptide regulatory hormone comprised of SEQ ID NO 1pG1u-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 involved in reproduction.Studies have shown that LHRH receptors are overexpressed in breast,prostate, endometrial and ovarian cancers, along with non-reproductivecancers, in comparison to normal cells, making the LHRH peptide a goodcandidate for drug targeting. See A. Qayum, et al., “The effects ofgonadotrophin releasing hormone analogues in prostate cancer aremediated through specific tumour receptors,” See British Journal OfCancer, 62 (1), pp. 96-9 (1990); M. Fekete, et al., “Characteristics anddistribution of receptors for [D-TRP6]-luteinizing hormone-releasinghormone, somatostatin, epidermal growth factor, and sex steroids in 500biopsy samples of human breast cancer,” Journal Of Clinical LaboratoryAnalysis, 3 (3), pp. 137-47 (1989); G. Emons, et al., “The use ofluteinizing hormone releasing hormone agonists and antagonists ingynecological cancers,” Hum Reprod, 9 (7), pp. 1364-79, (1994).

Additionally, LHRH has been shown to be effective as a stand-alonetreatment in certain cancer cases. Specific analogs of LHRH peptide foruse in targeting LHRH receptors in prostate and breast cancers are alsoknown in the art. See A. V. Schally, et al., “Peptide analogs in thetherapy of prostate cancer,” Prostate, 45, pp. 158-66 (2000); W. R.Miller, “Growth of human breast cancer cells inhibited by a luteinizinghormone-releasing hormone agonist,” Nature, 313, pp. 231-33 (1985); K.Szepashzi, et al., “Effective treatment of advanced estrogen-independentMXT mouse mammary cancers with targeted cytotoxic LHRH analogs,” BreastCancer Res. Treat., 56, pp. 267-76 (1999).

Existing methods for using hormones (such as LHRH and its analogs),peptides, antibodies and ligands to specifically target and directanticancer drugs and compounds to cancer cells via receptor bindingsites on those cells continue to be explored in field of biomedicalpharmacology with limited success. See Aggarwal, et al.,“[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugateinhibits pancreatic cancer cell growth in vitro and in vivo”International Journal of Cancer 2011, 129 (7), 1611-23; M. Wirth, etal., “Lectin-mediated drug delivery: influence of mucin on cytoadhesionof plant lectins in vitro,” Journal Of Controlled Release: OfficialJournal Of The Controlled Release Society, 79 (1-3), pp. 183-91 (2002);W. Mao, et al., “EphB2 as a therapeutic antibody drug target for thetreatment of colorectal cancer,” Cancer Research, 64 (3), pp. 781-8(2004); A. David, et al., “Design of a multivalent galactoside ligandfor selective targeting of HPMA copolymer-doxorubicin conjugates tohuman colon cancer cells,” Eur J Cancer, 40 (1), pp. 148-57 (2004); A.V. Schally, et al., “Cancer chemotherapy based on targeting of cytotoxicpeptide conjugates to their receptors on tumors,” European Journal OfEndocrinology/European Federation of Endocrine Societies, 141 (1), pp.1-14 (1999); and U.S. Pat. No. 9,221,877

The delivery of cisplatin by LHRH has been explored by the use ofnanogels. However, the direct linkage of LHRH to cisplatin as a methodof delivery has not been developed. The use of nanogels employs a morecomplex methodology involving cisplatin incorporation and release fromthe nanogel. Rendering it a less effective mode of delivery. See N.Nukolova, et al., “LHRH-Targeted Nanogels as a Delivery System forCisplatin to Ovarian Cancer,” Mol. Pharmaceutics, 10 (10), pp. 3913-3921(2013);

Therefore, there is a need for Platinum (Pt)-LHRH conjugates fortreating reproductive cancers that allows for the effective delivery ofcytotoxic drugs in the platinum family to tumor cells. Such a neededPlatinum (Pt)-LHRH conjugates would decrease adverse side effectsassociated with existing drugs, and limit nonspecific activity.

SUMMARY OF THE DISCLOSURE

The present invention describes the conjugation of luteinizinghormone-releasing hormone ([DLys⁶]-LHRH) with activated cisplatin usinga malonate linker that gives rise to new Platinum (Pt)-LHRH conjugatethat effectively targets tumor cells that express the LHRH receptor. Thepresent embodiment overcomes the existing shortcomings in this area byaccomplishing these critical objectives. The Pt-LHRH conjugate isintended to be used in the treatment of reproductive andnon-reproductive cancers expressing the LHRH receptor. The use of thenovel Pt-LHRH conjugate will increase the concentration of the platinumanticancer drug in tumor cells and attenuate unnecessary exposure tonormal cells. The new Pt-LHRH targeted anticancer drug has applicationsto prostate cancer and other metastatic cancers expressing the LHRHreceptor such as breast, melanoma, lung and pancreatic cancers,especially in late stage, highly invasive and aggressive stage IVtumors, and in reoccurring tumors as these are the most difficult totreat.

Other features and advantages of the present invention will becomeapparent from the following more detailed description, taken inconjunction with the accompanying drawings, which illustrate, by way ofexample, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a synthesis of a malonate (mal) linker in accordance witha preferred embodiment of the present invention;

FIG. 2 depicts the conjugation of SEQ ID NO 2 with activated cisplatinusing a malonate linker that gives rise to Pt-LHRH;

FIG. 3 depicts a mass spectrum of Pt-LHRH conjugate in accordance withthe preferred embodiment of the present invention;

FIG. 4 depicts a cytotoxicity of carboplatin, LHRH and Pt-LHRH againstprostate cancer cells in an methyl thiazol tetrazolium (MTT)proliferation assay in accordance with the preferred embodiment of thepresent invention;

FIG. 5 depicts a cytotoxicity of carboplatin, LHRH and Pt-LHRH againstbreast cancer cells in an MTT proliferation assay in accordance with thepreferred embodiment of the present invention;

FIG. 6 depicts a the cytotoxicity of Pt-LHRH against breast cancer cellsand normal mammary cells in an MTT proliferation assay in accordancewith the preferred embodiment of the present invention;

FIG. 7 compares drug uptake activity in breast cancer cells treated withcarboplatin and Pt-LHRH in accordance with the preferred embodiment ofthe present invention;

FIG. 8 compares drug uptake activity in prostate cancer cells treatedwith carboplatin and Pt-LHRH in accordance with the preferred embodimentof the present invention;

FIGS. 9A and 9B compare effects of Pt-LHRH, carboplatin and carboplatinwith LHRH on breast cancer cell migration in accordance with thepreferred embodiment of the present invention;

FIGS. 10A and 10B compares in-vivo effects of Pt-LHRH, carboplatin, andcarboplatin with LHRH against 4T1 breast cancer cells in female BALB/cmice in accordance with the preferred embodiment of the presentinvention;

FIG. 11 compares in-vivo effects of Pt-LHRH, carboplatin, andcarboplatin with LHRH on lung weight in female BALB/c mice implantedwith 4T1 breast cancer cells in accordance with the preferred embodimentof the present invention; and

FIG. 12 compares in-vivo effects of Pt-LHRH, carboplatin, andcarboplatin with LHRH on liver weight in female BALB/c mice implantedwith 4T1 breast cancer cells in accordance with the preferred embodimentof the present invention;

FIG. 13 depicts the conjugation of SEQ ID NO 2 with activated cisplatinusing a malonate linker in a PEGylated state that gives rise toPt-Peg-LHRH;

FIG. 14 depicts the conjugation of SEQ ID NO 2 with activated cisplatinusing a malonate linker with variation in linker length that gives riseto Pt-n-LHRH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following discussion that addresses a number of embodiments andapplications of the present invention, reference is made to theaccompanying drawings that form a part hereof, and show by way ofillustration specific embodiments in which the invention may bepracticed. It is to be understood that other embodiments may be utilizedand changes may be made without departing from the scope of the presentinvention.

Various inventive features are described below that can be usedindependently of one another or in combination with other features.However, any single inventive feature may not address any of theproblems discussed above or only address one of the problems discussedabove. Further, one or more of the problems discussed above may not befully addressed by any of the features described below.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise. As used herein, the term ‘about” means +/−5% of the recitedparameter. All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “wherein”, “whereas”, “above,” and“below” and words of similar import, when used in this application,shall refer to this application as a whole and not to any particularportions of the application.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While the specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

The present invention provides a compound and a method for generatingnew Platinum (Pt)-LHRH conjugate which will allow for the effectivedelivery of cytotoxic drugs in the platinum family to treat tumor cellswith decreased adverse side effects associated with existing drugs, andlimit nonselective activity. In a preferred embodiment, LHRH isconjugated to activated cisplatin using a malonate (mal) linker on[DLys6]-LHRH. The attachment of the mal linker on position 6 of the LHRHensures that the intrinsic properties of the LHRH peptide aremaintained. In the preferred embodiment, an ester bond is introducedbetween the components of the conjugate. Existing published reports haveshown that such a bond is hydrolyzed by cellular esterase, leaving thedrug free to act at the cellular level. See Aggarwal, et al.,“[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugateinhibits pancreatic cancer cell growth in vitro and in vivo”International Journal of Cancer 2011, 129 (7), 1611-23; U. S. Pat No:0263500A1.

In the preferred embodiment, LHRH is conjugated to activated cisplatinusing the mal linker without the additional use of Polyethylene glycol(PEG) groups or chains, ligands, peptides, hormones, or other linkers.The addition of a PEG group to a linker is known to provide syntheticcompound solubility or enhance bioavailability. In the preferredembodiment, the Pt-LHRH conjugate does not require a PEGylated linker tobe soluble and effective. Alternatively, in a further embodiment, thePt-LHRH conjugate may incorporate a PEGylated linker to enhancesolubility (see FIG. 14). PEGylation has been employed duringnanoparticle and NGR-Pt formulation. See M. Ndinguri, et al., “Peptidetargeting of platinum anti-cancer drugs,” Bioconjugate Chem., 20, pp.1869-78 (2009); N. Nukolova, “LHRH-targeted nanogels as delivery systemfor cisplatin to ovarian cancer,” Mol. Pharmaceutical, 10 (10), pp.3913-21 (2013); U.S. Patent Application No. 2011/0104074 A1.

In addition, surprisingly binding of unaltered cisplatin is unfavorableand activated cisplatin which was found to be needed for binding to themal-LHRH moiety. Further, the Pt-LHRH is highly soluble and has not beenshown to precipitate out of solution even after four months of storagein a 10 mM solution at 4° C. This could aid in therapeuticadministration of the compound.

The present invention describes the use of Boc protecting group insteadof Fmoc in the last amino acid (Boc-Glu(OtBu) (see. FIG. 2). This allowsfor a straight forward synthesis because it reduces the number ofsynthetic steps and avoids unwanted side chain reactions that can occurat the glutamic acid position. The acid labile Boc protecting group isconveniently removed at the end during cleavage of the peptide from theresin. The activated cisplatin is used to bind to the mal-LHRH moiety.The malonate linker chelates platinum in a similar manner tocarboplatin, even with variations in linker length, as shown in FIG. 13.As shown in FIG. 13, additional carbons may be added to the malonatelinker without altering the desired functionality or effect of Pt-LHRH.The end compound (see FIG. 2) chemically resembles carboplatin. Hence,carboplatin is utilized for treatment comparison. The Pt-LHRH has moreeffective systemic distribution and bioavailability to carboplatin. Inaddition, the cellular internalized into the cancer cell following itsPt-LHRH is cleaved by proteases which produces a platinum complex thatresembles cisplatin in structure and function.

In one embodiment, the mal linker may be utilized in combination withPEG groups, ligands, peptides, hormones, or other linkers.

In one embodiment, cisplatin may be substituted with any other cytotoxicdrug in the platinum family or any known derivatives thereof.

In one embodiment, aliphatic linkers other than the mal linker may beused, either alone or in combination with other linkers.

Finally, in yet another embodiment, the targeting peptide can beendogenous LHRH or an analog thereof, including but not limited to,[D-Trp6]-LHRH, [D-Ala6]-LHRH, [G1n8]-LHRH, luteinizing hormone,chorionic gonadotropin, antide, gonadorelin, leuprolide, leuprorelin,histerlin, buserelin, triptorelin, and fragmented portions containing abinding moiety. In addition, analogs for LHRH, including LHRH receptoragonists and antagonists may be used in alternate embodiments. See A. V.Schally, et al., “Peptide analogs in the therapy of prostate cancer,”Prostate, 45, pp. 158-66 (2000); W. R. Miller, “Growth of human breastcancer cells inhibited by a luteinizing hormone-releasing hormoneagonist,” Nature, 313, pp. 231-33 (1985); K. Szepashzi, et al.,“Effective treatment of advanced estrogen-independent MXT mouse mammarycancers with targeted cytotoxic LH-RH analogs,” Breast Cancer Res.Treat., 56, pp. 267-76 (1999).

The novel Pt-LHRH conjugate is intended to be used in the treatment ofreproductive and non-reproductive cancers expressing the LHRH receptor.The use of the novel Pt-LHRH conjugate will increase the concentrationof the platinum anticancer drug in tumor cells and attenuate unnecessaryexposure to normal cells. The new Pt-LHRH targeted anticancer drug hasapplications to prostate cancer and other metastatic cancers such asbreast, melanoma, lung and pancreatic cancers, especially in late stage,highly invasive and aggressive stage IV tumors, and in reoccurringtumors as these are the most difficult to treat.

In one embodiment the Pt-LHRH conjugate targets and binds to the LHRHreceptors of any cancer cell or any tumor microenvironment thatexpresses these receptors. Moreover, protease produced by the tumorcells effectively cleave the linker to release the “warhead,” i.e.,cisplatin, carboplatin, or other platinum drug or derivative, andthereby to achieve more effective concentrations of the therapeuticagents in the targeted area. Further, the targeted Pt-LHRH conjugatewill selectively kill tumor cells and prevent or reduce dose-limitingsystemic toxicity of said chemotherapeutic drugs.

The following examples illustrate the innovation and significance ofusing Pt-LHRH are discussed in studies involving prostate and breastcancer, however, the applications of, mechanisms of action and/orrelevance of Pt-LHRH are not restrictive to treatment of those cancers.

Example 1

FIG. 1 depicts the synthesis of Mal Linker for conjugation with the LHRHPeptide and cisplatin in accordance with a preferred embodiment of thepresent invention. In the preferred example, the mal linker wassuccessfully prepared by alkylating di-tert-butyl malonate usingbromopropylphtalimide using a strong base under absolute dry conditions(see FIG. 1(A)). The product obtained, di-tert-butyl2-(3-phthalimidopropyl) malonate was isolated using silica columnchromatography and deprotected using hydrazine in absolute ethanol togive a free primary amine end (see FIG. 1(B)). The primary amine wasreacted with gluatric anhydride to give di-tert-butyl2-(3-(4-Carboxybutanamido)propyl)-malonate (Mal) (see FIG. 1(C)) asdescribed by M. Ndinguri, et al., Bioconjugate Chem., 20, pp. 1869-78(2009) thereby introducing a carboxylic functional group which wasattached to the peptide moiety. Each step was purified by columnchromatography or extraction and dried before proceeding to the nextstep.

Example 2

FIG. 2 depicts a synthesis of Pt-LHRH conjugate in accordance with apreferred embodiment of the present invention. The Mal-LHRH decapeptidewas synthesized using Fmoc solid phase chemistry techniques and thenchelated to activated cisplatin. Fmoc-Rink Amide-AM resin was placedonto a reaction vessel and washed with DMF and DCM in continuous-flowmode using a PS3 peptide synthesizer. The side chain protected aminoacids derivatives used were Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)—OH,Fmoc-Leu-OH, Fmoc-DLys(Aloc)—OH, Fmoc-Tyr(tBu)—OH, Fmoc-Ser(tBu)—OH,Fmoc-Trp(Boc)—OH, Fmoc-His(Trt)—OH and Boc-Glu(OtBu)—OH. Moreover, theuse of Boc protecting group instead of Fmoc in the last amino acid(Boc-Glu(OtBu) allows for a straightforward synthesis because it reducesthe number of synthetic steps and avoids unwanted side chain reactions.The acid labile Boc protecting group is conveniently removed in the endduring cleavage of the peptide from the resin. The amino acid couplingsemployed four equivalents of amino acid and (2-(6-Chloro-1H-benzotriazole-1-yl)-1, 1,3,3-tetramethylaminium hexafluorophosphate(HCTU) and sometimes (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyAOP) or other knowncoupling reagents dissolved in 0.4 M N-methylmorpholine (NMM) in DMF atroom temperature. The allyloxycarbonyl (aloc) protecting group on theD-lys was selectively removed using palladium. The amine end of theD-lys was coupled with the mal linker (see FIG. 2) and the conjugatecleaved from the resin using TFA-Tips-H2O-Phenol or TFA-Tips-H2O orTFA-Thioanisole-H2O or TFA-Thioanisole-H2O cocktail to yield ofLHRH-mal. Activated cisplatin was formed using cisplatin, silver nitrateand water in accordance with known methods as described inBerners-Price, et al., “Hydrolysis products of cisplatin: pKadeterminations via [1H, 15N] NMR spectroscopy,” Journal of the ChemicalSociety, Chemical Communications (1992) 789-791; Appleton et al., “NMRstudy of acid-base equilibria and other reactions of ammineplatinumcomplexes with aqua and hydroxo ligands,” Inorganic Chemistry, (1989)28, 1989-1993. Reaction of LHRH-mal and activated cisplatin yieldedPt-LHRH. Conjugation of Pt to LHRH yields a structure similar tocarboplatin no matter the malonate linker carbon chain length (see FIG.13) nor the PEGylation state (see FIG. 14).

FIG. 3 depicts a mass spectrum of Pt-LHRH conjugate in accordance with apreferred embodiment of the present invention.

Example 3

Cytotoxicity Assay, in-vitro assays were used to explore the role ofPt-LHRH conjugates in cytotoxicity. The cytotoxic effect of freecarboplatin, free LHRH and Pt-LHRH conjugate were assayed and comparedusing methyl thiazol tetrazolium (MTT)(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay asdescribed in S. Aggarwal, et al., “Inhibition of growth and survival ofhuman head and neck squamous cell carcinoma cells by curcumin viamodulation of nuclear factor-KB signaling,” International Journal ofCancer, 111 (5), pp. 679-92 (2004).

Cells from prostate cancer cell line PC-3 (CRL-1435) and breast cancercell line 4T1 (CRL-2539) were obtained from American Culture TypeCollection (ATCC) and cultured in DMEM supplemented with 10% FBS, 100units/mL penicillin, and 100 μg/mL streptomycin. Two thousand cells ofPC-3 or 4T1 were treated with various concentrations of freecarboplatin, LHRH or Pt-LHRH for 24 hours followed by further incubation(no treatment) for 48 hours. The results were assayed using MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.Pt-LHRH showed a significant increase in cytotoxicity compared tocarboplatin in both cell lines. As shown in FIG. 4 and FIG. 5, stage IVprostate and breast cancer cells showed a significant decrease in cancercell proliferation/viability compared to carboplatin alone.

As shown in FIG. 6, Pt-LHRH attenuates 4T1 breast cancer cell viabilitycompared to normal mammary cells (3T3). The 3T3 cells were obtained fromATCC (CRL-2752) were treated with Pt-LHRH from a range of 0.1 μM to 100μM. The viability rates were analyzed by a MTT assay as previouslydescribed. As shown in FIG. 6, there was a significant decrease innormal mammary cell cytotoxicity compared to 4T1 cancer cells whentreated with Pt-LHRH. These results indicate that the targeting activityof Pt-LHRH is effective and suggest that there may be less deleteriousside effects from treatment with Pt-LHRH as reported from cisplatin orcarboplatin alone.

Example 4

Drug Uptake Measurements

4T1 Breast Cancer cells were treated with a 100 μM concentrationcarboplatin or Pt-LHRH for 24 hours. The collected samples were lysedwith nitric acid and analyzed for the presence of platinum usinginductive coupled plasma (ICP-OES). As shown in FIG. 7, there was asignificant increase in uptake (20 fold) of Pt-LHRH compared tocarboplatin.

PC-3 Prostate Cancer cells were treated with a 100 μM concentration ofcarboplatin or Pt-LHRH for 24 hours. The collected samples were lysedwith nitric acid and analyzed for the presence of platinum usinginductive coupled plasma (ICP-OES). As shown in FIG. 8, there was asignificant increase in uptake (20 fold) of Pt-LHRH compared tocarboplatin.

Example 5

Migration Assay

Migration of 4T1 Breast Cancer cells was examined using a scratch/woundhealing assay and a transwell assay. For the scratch assay, the 4T1cells were cultured in a 6-well plate until a confluent monolayer wasformed. A 20 μl pipet tip was used to scratch the wells. The wells weresequentially rinsed with PBS and cultured in DMEM supplemented with 10%FBS. The wells were treated with PBS, carboplatin (100 μM), LHRH (100μM), or Pt-LHRH (100 μM). Four representative 10×images were taken at 0,6, and 24 hours and the gap width was quantified using an average ofthree leading edge measurements for each image. As shown in FIGS. 9A and9B, there was a significant decrease in 4T1 cell migration duringPt-LHRH compared to carboplatin treatment. These results indicate thatPt-LHRH may be more effective at inhibiting metastasis of breast cancercells than carboplatin alone.

Example 6

In-vivo Analysis of Impact of Pt-LHRH Conjugate on Tumor Growth

In this experiment at least one eleven week old female Balb/c mice wereutilized. The animals were allowed to acclimate for 1 week beforeexperimentation and were maintained on a 12 hours light and dark cycle,and fed standard rodent chow (Prolab ISOPRO RMH 3000 Irradiated LabDiet; Purina Mills International). Animal protocols were approved by thecommittee on animal research care and use at Eastern KentuckyUniversity. 4T1 tumors were grown in the right abdominal mammary fat padof the mice for 7 days. After tumor initiation (˜100 mm3) the mice wereadministered Pt-LHRH (carboplatin molar-equivalent dosage) through IPinjection and compared to those treated with carboplatin (5 mg/kg/wk),no treatment, and LHRH plus carboplatin (unbound) controls for 2 weeks.Tumor growth was monitored daily and tumor volumes (mm3) were calculatedusing the formula: (width) 2×length/2, where width is the smaller of thetwo measurements. At two weeks the mice were sacrificed and tumor volumeand weight were measured.

As shown in FIGS. 10A and 10B, Pt-LHRH significantly attenuated tumorgrowth compared to control. Importantly at this low dose, Pt-LHRH wasfound therapeutically effective, whereas, no statistically significantreduction in tumor growth was found with carboplatin treatment. Previouspublications highlight the use of higher treatment doses of carboplatin(ex. 25 mg/kg/wk) needed to mediate a significant therapeutic effect ontumor growth. J. M. Scalici, et al., “Imaging VCAM-1 as an indicator oftreatment efficacy in metastatic ovarian cancer,” J. Nucl. Med., 54(11), pp. 1883-9 (2013); B. Das, et al., “Squalene selectively protectsmouse bone marrow progenitors against cisplatin and carboplatin-inducedcytotoxicity in vivo without protecting tumor growth,” Neoplasia, 10(10), pp. 1105-19 (2008); O. Karginova, et al., “Efficacy of carboplatinalone and in combination with ABT888 in intracranial murine models ofBRCA-mutated and BRCA-wild-type triple-negative breast cancer,”Molecular Cancer Therapeutics, 14 (4), pp. 920-30 (2015); X. M. Li, etal., “Preclinical relevance of dosing time for the therapeutic index ofgemcitabine-cisplatin,” British Journal of Cancer, 92 (9), pp. 1684-9(2005). Additionally, as shown in FIG. 11, there was a slight decreasein lung weight and a significant decrease in the number of lung tumorlesions in the Pt-LHRH treated mice indicating that Pt-LHRH mayattenuate breast cancer lung metastasis. Additionally, there was nodifference in liver weight (see FIG. 12). Observably, there was normallocomotor activity exhibited by the mice treated with Pt-LHRH comparedto the control and carboplatin treated groups. This observation may be aresult from not only cancer regression, but decreased systemic toxicity(side effects).

Pt-LHRH is projected to be used in clinical settings to targetreproductive cancers and any cancer type expressing the LHRH receptor inboth animals and humans.

A compound that is a “conjugate” of two domains refers to a compound inwhich the two domains (or moieties) are covalently bonded to oneanother, either directly or via a linker.

Compounds used in the present invention may be administered to a patientby any “effective route”. Said effective route may include oral,intrapulmonary, parenteral, subcutaneous, intramuscular, intravenous,intra-arterial, intrathecal, transdermal, and via mucous membrane(nasal, sublingual, rectal, urinary, and reproductive tract). Parenteralinfusions can include intraperitoneal administration. The compounds arecapable of transdermal delivery in the form of a slow-releasesubcutaneous implant.

Compounds used in the present invention may be administered by anypharmaceutically acceptable carrier preparations. The carriers andpreparations may include sterile, aqueous or non-aqueous solutions,suspensions, and emulsions. Compounds may be administered in combinationwith a slow-release mechanism. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. The active therapeutic ingredient may be mixedwith excipients that are pharmaceutically acceptable and are compatiblewith the active ingredient. Suitable excipients include water, saline,dextrose, glycerol and ethanol, or combinations thereof. Intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers, such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present such asantimicrobials, anti-oxidants, chelating agents, inert gases, and thelike.

The form may vary depending upon the route of administration. Forexample, compositions for injection may be provided in the form of anampoule, each containing a unit dose amount, or in the form of acontainer containing multiple doses.

A compound in accordance with the present invention may be formulatedinto therapeutic compositions as pharmaceutically acceptable salts.These salts include acid addition salts formed with inorganic acids, forexample hydrochloric or phosphoric acid, or organic acids, such asacetic oxalic, or tartaric acid and the like. Salts also include thoseformed from inorganic bases such as sodium, potassium, ammonium, calciumor ferric hydroxides, and organic bases such as isopropylamine,trimethylamine, histidine, procaine and the like.

As used herein, an “effective amount” of a compound is an amount thatwhen administered to a patient inhibits or reduces the growth oftargeted tumors to a clinically significant degree; or alternatively, toa statistically significant degree as compared to control.

As used herein, a “platinum drug” is a chemotherapeutic agent in theplatinum family and includes carboplatin, cisplatin, oxaliplatin and anyderivatives thereof. A platinum drug may also refer to the Pt groupmetal, alone.

As used herein, a “linker” refers to any linkers or combination oflinkers known in the art including acetate linkers, PEG groups orvariations in carbon length chains, ester linkers, sugars, lectins,antibodies and their fragments, hormones and hormone analogues.

As used herein, an “analog” is a molecule comprised of a peptidesequence that is comparable to the endogenously produced compound, inwhich, one or more residues have been replaced, deleted or modified withan alternate desired moiety. An analog of LHRH can be a natural or asynthetic peptide that resembles LHRH in structure and/or function.

All the references cited in this disclosure are hereby incorporated byreference in their entirety.

The foregoing description of several embodiments of the Pt-LHRHconjugate and treatment methods utilizing the Pt-LHRH conjugate has beenpresented for purposes of illustration. It is not intended to beexhaustive or to limit the application to the precise forms disclosed,and obviously, many modifications and variations are possible in lightof the above teaching. It is understood that the invention may beapplied in ways other than as specifically set forth herein withoutdeparting from the scope of the invention.

This application incorporates by reference the sequence listingsubmitted electronically as an ASCII text file on Apr. 2, 2018, titled“Sequence Listing LHRH Platinum Conjugates,” which was submitted withU.S. patent application Ser. No. 15/367,428 filed Dec. 2, 2016, andincorporated by reference herein.

1-14. (canceled)
 15. A method for delivering a cytotoxicchemotherapeutic agent to a cell that expresses a lutenizing hormonereleasing hormone (LHRH) receptor comprising administering to the cell acompound comprised of: a LHRH or analog thereof; a cytotoxic platinumchemotherapeutic agent; and a linker, wherein the linker comprisesmalonate; wherein the LHRH or analog thereof is covalently bonded to thecytotoxic platinum chemotherapeutic agent utilizing the linker andfurther wherein the LHRH analog is selected from the group consisting of[DLys⁶]-LHRH, [D-Trp6]-LHRH, [D-Ala6]-LHRH, [GIn8]-LHRH, antide,gonadorelin, leuprolide, leuprorelin, histerlin, buserelin, ortriptorelin.
 16. The method of claim 15, wherein the cell is a cancercell.
 17. The method of claim 16, wherein the cancer cell is a prostatecell or a breast cell.
 18. The method of claim 16, wherein the cell isin vivo.
 19. The method of claim 15, wherein the cell is in a cancerpatient.
 20. The method of claim 19, wherein the compound selectivelykills cancer cells to allow for reduced dosing of the cytotoxic platinumchemotherapeutic agent.
 21. The method of claim 15, wherein the compoundis administered via oral, intrapulmonary, parenteral, subcutaneous,intramuscular, intravenous, intra-arterial, intrathecal, transdermal, ormucous membrane route.
 22. The method of claim 15, wherein the compoundis administered intravenously.
 23. The method of claim 15, wherein thecompound further comprises at least one of a pharmaceutically acceptablecarrier, an excipient, a vehicle, or a combination thereof.
 24. Themethod of claim 15, wherein the linker further comprises a polyethyleneglycol (PEG) group.
 25. The method of claim 15, wherein the linkercomprises malonate and an additional carbon.
 26. The method of claim 15,wherein the linker further comprises an additional carbon.
 27. Themethod of claim 15, wherein the cytotoxic platinum chemotherapeuticagent comprises cisplatin.
 28. The method of claim 15, wherein thecytotoxic platinum chemotherapeutic agent comprises carboplatin.
 29. Themethod of claim 15, wherein the comprises the amino acid sequence as setforth in SEQ ID NO:
 2. 30. The method of claim 15, wherein the LHRHcomprises the amino acid sequence as set forth in SEQ ID NO: I.
 31. Themethod of claim 15, wherein the linker does not include a polyethyleneglycol (PEG) group.
 32. The method of claim 15, wherein the comprises anamino terminal pyroglutamic acid.
 33. A method for delivering acytotoxic chemotherapeutic agent to cells in a subject that expresslutenizing hormone releasing hormone (LHRH) receptor, the methodcomprising administering to a subject a compound comprised of: a LHRH oranalog thereof; a cytotoxic platinum chemotherapeutic agent; and alinker, wherein the linker comprises malonate; wherein the LHRH oranalog thereof is covalently bonded to the cytotoxic platinumchemotherapeutic agent utilizing the linker and further wherein the LHRHanalog is selected from the group consisting of [DLys⁶]-LHRH,[D-Trp6]-LHRH, [D-Ala6]-LHRH, [GIn8]-LHRH, antide, gonadorelin,leuprolide, leuprorelin, histerlin, buserelin, or triptorelin.
 34. Themethod of claim 33, wherein the subject is a cancer patient.